DESCRIPTION: There is a large body of evidence that suggests that the unique extracellular environment in the brain, which contains few fibrous proteins and high amounts of hyaluronic acid (HA), is responsible for the characteristic aggressiveness and resistance to conventional treatments observed in brain tumors categorized as glioblastoma mulitforme (GBM). However the specific interactions between tumor cells and the extracellular matrix (ECM) and their relationship to tumor physiology are largely unknown. In general, these mechanisms have been challenging to elucidate, in large part because of the lack of physiologically translatable models that can be studied in controlled context ex vivo. To fulfill this need, we propose a biomaterials-based approach to create three-dimensional (3D) cultures of primary GBM cells that accurately represent the complex, in vivo microenvironment and preserve physiology of clinical tumors. These culture platforms permit independent, orthogonal control of multiple micro environmental parameters (modulus, peptide content, HA content) with unprecedented precision. By combining this approach with dynamic measurements of micro environmental parameters and transcription factor (TF) activity, we propose to investigate the relationships between the GBM microenvironment and drug resistance on multiple biological levels. In particular, we aim to identify specific cell-ECM interactions as potential clinical targets whose disruption inhibits acquisition of resistance epidermal growth factor receptor (EGFR) inhibitors. Aim 1 of this application describes a plan to 1) fully characterize the biochemical and biophysical landscape in clinical GBM and 2) identify specific cell-ECM interactions that relay these microenvironment cues, inducing GBM cells to exhibit poor response to treatment with EGFR inhibitors. To identify specific interactions, patient- derived GBM cells will be cultured within 3D, HA-based hydrogels, which represent a controlled experimental system where HA concentration, density and identity of available integrin-binding peptides, and mechanical modulus can be precisely varied and their independent effects distinguished. In parallel, Aim 2 describes studies in which the intracellular response to the EGFR inhibition using a high-throughput approach to dynamically monitor TF activity in live, 3D cultures. As the unique ECM in the brain is a major mediator of drug resistance, the brain-mimetic hydrogel platforms outlined in Aim 1 will provide an ideal culture environment in which to perform these studies. These experiments will characterize the complex transcriptional events that occur when non-resistance GBM cells are first exposed to EGFR inhibitors and quantitative measure the progressive response to these treatments, including acquisition of resistance, in real-time. Together, these studies provide a promising opportunity to identify new pharmacological targets for adjunct treatments to EGFR inhibition at multiple biological levels (i.e., cell surface-ECM interface in Aim 1 and TF binding to gene promoters in the nucleus in Aim 2). Given the significant heterogeneity of cells within GBM tumors and the variety of mechanisms that these cells may adopt to gain resistance to chemotherapeutic drugs [46,47,50,51], a systems-level understanding of the signaling networks involved would be a valuable asset to the field for identifying potent pharmacological targets for GBM treatment.